Brainstem noradrenergic system depression by cyclobenzaprine

.Vp,,ropkilnr,u~~oi~,~?. Vol. 19. pp ??I 1” 224 Pcrgamon Pros Ltd IYXO Pnntcd m Great Bntaln

BRAINSTEM

NORADRENERGIC SYSTEM BY CYCLOBENZAPRINE

DEPRESSION

C. D. BARNES,S. J. FLINGand J. GINTAUTAS Department

of Physiology, Texas Tech University Lubbock. TX 79430. U.S.A. (Accrprrd

School

of Medicine,

25 July 1979)

Summary-A possible mechanism for CBZ’s depressant action on the brainstem noradrenergic system was investigated in precollicularly decerebrated cats immobilized with Flaxedil (2 mg/kg, i.v.). Phenoxybenzamine (2 mg/kg, i.v.) was consistently observed to antagonize CBZ’s suppression of both the flexor and extensor MSRs. Subsequent evidence for the brainstem noradrenergic system mediating the CBZ effect was established by extracellular recordings. Unitary discharges of LC neurons identified histologically were demonstrated to be inhibited chiefly by CBZ (I mg.‘kg. i.v.). In some instances. the inhibition of spontaneous coerulear activity was found to parallel temporally the MSR response following CBZ administration. Coerulospinal neurons characterized by physiological cri!eria also revealed diminished activities following CBZ (0.5 mg/kg, i.v.). A decreased neuronal somatic excitability was also observed by the antidromically driven coerulospinal neurons following CBZ. It is concluded that CBZ’s depressant action is mediated. at least in oart. bv, the noradrenergic coerulospinal system. Other possible mechanisms of CBZ action are also discussed.

A number of lines of evidence suggests that one of the main sites of action of cyclobenzaprine (CBZ) in its effect as a skeletal muscle relaxant is on neurons of the brainstem reticular formation. This evidence includes both reflex and single cell recording from the spinal cord (Share and McFarlane, 1975; Barnes, 1976) and the brainstem (Barnes, 1976; Barnes and Adams, 1978). Recent evidence of pontine reticular groups which are essentially involved in extensor muscle tone and sensitive to cholinergic drugs (Pompeiano and Hoshino, 1976) is encouraging in the light of CBZ’s action on cholinergic systems. The reported effects of CBZ are reduction of the extensor monosynaptic reflex (MSR) and also, but to a lesser degree, the flexor MSR. For this to be due to disfacilitation, as proposed (Barnes, 1976). a descending system which tonically facilitates both flexors and extensors would be required. The literature contains very few references to support the existence of such systems in the brainstem. Early studies on the basal ganglia (Stern and Ward, 1962) do report such an influence from higher centers, however, and the more recent work by York (1972, 1973) indicates that stimulation of the substantia nigra facilitated both flexor and extensor motoneuron pools. This was also encouraging in that CBZ has been reported to be effective in treating Parkinson’s disease (MolinaNegro and Illingworth, 1973), a disorder known to be linked with the basal ganglia in general and the substantia nigra in particular. Recent work in this laboratory (Strahlendorf, Strahlendorf, Kingsley, Gintautas and Barnes, 1979) determined that stimulation in the area of the locus coeruleus (LC) produces both extensor and flexor facilitation in the decerebrate cat. That noradrenergic neurons were responsible for the effect was demon-

strated by the blockade with phenoxybenzamine (PBZ). Furthermore, direct projections from LC to spinal ventral columns has been reported (Nygren and Olsen, 1977; Commissiong, Hellstrijm and Neff, 1978). The present series of experiments was designed to determine whether the brainstem site which is facilitatory to both extensors and flexors was also affected by CBZ. METHODS

Cats of either sex, decerebrated at the precollicular level under ether anesthesia, were used as subjects. After brainstem transection, the ether was discontinued and no data were taken for at least three hours. The femoral, obturator, and hamstring nerve trunks on both sides were cut. All animals were subjected to lumbar laminectomy removing the dorsal aspect of the Vth, VIth, and VIIth lumbar vertebrae to expose the VIth and VIIth lumbar roots and then placed in a stereotaxic head-holder with their spines immobilized by clamping the dorsal processes of L3 and L4. The popliteal fossa of the left leg was opened and the gastrocnemius-soleus (GS) and the common peroneal (CP) branches exposed, crushed distally, and placed on pairs of electrodes. The monosynaptic reflex (MSR) in the lumbar cord was monitored by either stimulating the central end of the cut dorsal root of left L7 and recording the response peripherally from both extensor and flexor nerves simultaneously, or alternating the peripheral stimulation and recording both pools from the central end of the cut left L7 ventral root. In either case, the stimulus was a 0.1 msec rectangular pulse delivered through a photon coupled stimulus isolator at a repetition interval of 4 sec. 221

222

C‘. D. HAKWS,S. J. FLJNC;and J. GINTALJTAS

The various brainstem sites were stimulated singularly or with a train of four 0.1 msec rectangular pulses at 100 Hz through concentric bipolar electrodes with 0.5 mm tip separation. The electrodes were placed stereotaxically and their position verified histologically. Single cell recordings were made using stainless steel microelectrodes or glass microelectrodes filled with 2 M NaCl and saturated with pontamine sky blue. To antidromically activate LC neurons. a single 0.1 msec rectangular pulse was delivered to the ipsilateral ventral horn of L7 through concentric bipolar electrodes with 0.5 mm tip separation The electrodes were placed under visual control and their position verified histologically. All exposed neural tissue was covered with warm mineral oil. The animals’ temperatures were maintained at 37 & 1 C. Further tmmobilization was achieved by repeated doses of Flaxedil (gallamine triethiodide. 2 mg/kg) as needed and the animal maintained by artificial ventilation. Arterial blood pressure was recorded continuously from a catheter in the right femoral artery. Drugs were delivered into the inferior vena C;~V;Ithrough a catheter inserted into the right femoral vein.

C

RESULTS Cyclobenzaprine was given in a series of 46 animals in a dose of 0.5 or 1 mg/kg to determine the amount of MSR depression, which on the average was 55”,,. Two hours after the initial dose of CBZ and following the MSR return to within 15”,, of the pre-drug level, a 2 mgkg dose of PBZ was given. Records were taken as soon as the preparation was stable. A second dose of CBZ was then given and records taken a third time. Following PBZ. there was always a decrease in the amplitude of the MSR that averaged 500,,. The following doses of CBZ produced little or no further depression, on the average 3”1, (Fig. 1). To guard against the dependence of the PBZ effects on a previous injection of CBZ, 12 animals were given PBZ as ;I first drug followed. after stability was regained. by 1 mg,ikg of CBZ. The results were similar to the previous findings with the PBZ-induced depression averaging 60”,, and the following CBZ producing an additional 2’?,,. A total of 21 spontaneously active neurons were recorded in the area of the locus coeruleus before and after the administration of CBZ. Of these cells. 16 were found to decrease their discharge rate following

CBZ

PBZ

CBZ

Fig. I. Effectiveness 01‘ phcnoxybenramine MSR. Top, C: Control MSR before any after initial CBZ. PBZ: 20 min after 2.0 second I.0 m&kg CBZ which was given taken over a 6.7

(PBZ) in blocking CBZ’s depression of the gastrocnemius drugs given. CBZ: IO min after l.Omg,‘kg CBZ. Middle, C: 2 hr mg,!kg PBZ. Bottom. C: 40 min after PBZ. CBZ: 10 min after a immediately following C. Each trace is 100 consecutive MSRs min period. Calibration: 20 msec. I mV.

Cyclobenzaprine

223

and NE neurons

Fig. 2. Effect of CBZ on cell in area of locus coeruleus. C: control recordings before any drug. CBZ: IO min following 1.0 mg;kg cyclobenzaprine. SA: spontaneous activity. GS: cell response to single shock delivered to contralateral gastrocnemiussoleus nerve.

1 mg/‘kg of CBZ (Fig. 2) while two showed no response and three demonstrated an increased rate. The amount of decrease varied widely but was always at least 25% and in three cases, the cell was completely silenced. In all cases where the MSR was also being recorded, the cell response to CBZ followed very closely in time the MSR response. Subsequent histological examination indicated that those cells which increased their discharge rate during CBZ, though small in number, all resided above the LC in the periaqueductal gray. Two of the cells which decreased their discharge rate were located below the LC in the nucleus reticularis pontis oralis and the remaining cells were located in LC. In a group of six cells, physiologically identified as LC neurons by their slow tonic discharge rate and burst response to nociceptive stimulation, attempts were made to identify them as the origin of long descending fibers. After identifying the LC cell, a stimulus was applied to the ventral horn of ipsilateral L7. If the LC cell responded, the stimulus frequency was

increased to > 100 Hz to differentiate between transsynaptic and antidromic activation. Three of the cells could not follow 10 Hz, had an irregular delay and classified as transsynaptic. The were, therefore, remaining three cells followed > 100 Hz with a constant delay and were thus classified as antidromic (Fig. 3). This was further verified by collision with spontaneous discharges. All six cells decreased their spontaneous discharge rate following 0.5 mg!kg CBZ. In all three antidromically activated neurons, though the conduction velocity remained unchanged. as indicated by the time from shock artifact to the initial phase

of the

somato-dendritic

the amplitude

action

potential,

action

potential

the

rise was

time increased

the

and

decreased. DISCUSSION

The results of the present investigation support the notion that CBZ acts in part by depressing noradrenergic cells in the LC, which, as a final effect,

CBZ

0

Cl+

of

0531796

Fig. 3. Effect of CBZ on locus coeruleus cell with lcng descending axon. Column C: control recordings before any drug. Column CBZ: 15--l 8 min after 0.5 mg/kg of cyclobenzaprine. A: spontaneous activity. B: cell response to pinching between the toes of the right hind foot during the period of the underlining bar. C: 5 superimposed traces of the cell being activated antidromically by a 0. I msec, 100 /IA stimulus applied to the left ventral horn of L7. Calibration: A and B. 2 sec. 200 /IV; C. 2 msec, 100 /IV.

C. 0. BAKWS. S. J. F~INC; and J. GINTAL,TAS

224

tonically facilitates alpha motoneurons in the lumbar cord. Previous findings demonstrating that CBZ was equally effective with atropine in blocking brainsteminduced, physostigmine-enhanced. spinal cord inhibition (Barnes, 1976) led to the speculation that CBZ’s cholinergic blocking properties (Hughes, Lemons and Barnes, 1978) were responsible for its action. Additionally, Pompeiano and Hoshino (1976) reported that cells in the area of the LC were inhibited by the injection of physostigmine. Their explanation for the observed inhibition involved a functional interaction between monoaminergic neurons in the LC, which are inhibitory to gigantocellular tegmental field cholinergic neurons. which in turn are excitatory to the LC. It was proposed that this mechanism was involved in the cataplectic episode attending REM sleep. Though indeed a cholinergic block of tonic driving could be part of the effective action of CBZ. the involvement of brainstem noradrenergic neurons adds an additional factor. Cyclobenraprine is a tricyclic compound and 21s such has many properties similar to tricyclic antidepressants (Hughes (‘r trl., 197X). In a recent study, Svensson and Usdin (197X) demonstrated that tricyclic antidepressants inhibit noradrenergic neurons in the area of LC through a feedback system involving presynaptic r-receptors. Thus, CBZ may not only be blocking cholinergic facilitatory input, but also acting directly on the coerulear neurons to depress them through adrencrgic receptors. Inasmuch as the predominant reported action of iontophoretically applied norepinephrinc is inhibition on spinal interneurons (Engberg and Ryall, 1966) and motoneurons (Engberg and Marshall. I97 I ). this noradrenergic system may be acting at the lower brainstem or spinal cord by inhibiting a tonically active inhibitory pathway resulting in MSR disinhibition which would be withdrawn by C’BZ. Though possible, the authors do not favor this interpretation. The

concept

of a descending

noradrenergic

system

than inhibitory is against the general concept; however, it has been demonstrated that electrical stimulation in the area of Lc’ produces facilitation of both the flexor and extensor MSR in the lumbar cord and that this facilitation is diminished by a-adrenergic blocking agents (Strahlendorf (‘I u/., 1979). Additionally. the observation that the noradrenergic receptor stimulator. clonidine. releases a spinal neuronal network generating locomotion in spinal cats (Forssberg and Grillner. 1973) is consistent with 21 descending facilitatory effect. being

This present

facilitatory

rather

interpretation findings

is further that

strengthened

LC neurons,

which

by are

the anti-

dromically driven from the ventral horn of the lumbar cord, respond to CBZ by a decrease in spontaneous discharge rate and an increase in somatic action potential rise time. .~~,~flo,~,/~,~l~/~,~~t~,~lr.\~~~~This research *as supported by the Tarbox Parkinson’s Disease Institute and Merck Frosst Laboratories.

REFERENCES

Barnes, C. D. (1976). Effects of cyclobenzaprine on brainstem motor systems. N~wrophurmcudogy 15: 643-652. Barnes, C. D. and Adams. W. L. (1978). Effects of cyclobenzaprine on interneurons of the spinal cord. Neuropharmcrcoloyy 17: 445450. Ccmmissiong. J. W.. Hellstrijm. S. 0. and Neff, N. H. (197X). A new proJcction from locus coeruleus to the spinal ventral columns: histochemical and biochemical evidence. Brtrin Rv.s. 148: 207 2 13. Engberg. I. and Marshall. K. C. (1971). Mechanism of noradrenaline hyperpolariration in spinal cord motoneurons of the cat. ,4cr(1 ph~.sio/. sctrr~d. 83: 132~144. Engberg. 1. and Rqall, R. W. (1966). The inhibitor) action of noradrenaline and other monoamines on spinal neurons. J. Physid.. Loud. 185: 29X- 321. Forssberg. H. and Grillner. S. (1973). The locomotion of the acute spinal cat injected with clonidine iv. Bruin Rr. 50: 184 186. Hughes, M. J., Lemons. S. and Barnes, C. D. (1978). Cqclobcnzaprine: some pharmacological cardiac actions. Lifi Sci. 23: 2779~2786. Molina-Negro, P. and Illingworth. R. A. (1973). Rapport preliminaire sur I’action de la cyclobenzaprine (MK-130) dnns la maladie de Parkinson. L’,lio,r ,Mcrl. Cot]. 102: 303 30x. Nygren, L. and Olsen, L. (1977). A new major projection from locus coeruleus: the main source of noradrenergic nerve terminals in the ventral and dorsal columns of the spinal cord. Brtrlu Rrs. 132: 85 93. Pompeiano. 0. and Hoshino. K. (1976). Central control of posture: reciprocal discharge by two pontinc neuronal groups leading to suppression of decerebrate rigidity. Brtrill Rex 116: I .3I 138. Share, N. N. and McFarlane. c‘. S. (1975). Cyclobenzaprmc: A novel centrally acting skeletal muscle relaxant. Nc,uroykur,~lu~olog~ 14: 675%684. Stern. J. and Ward, A. A. (1962). Supraspinal and drug modulation of the alpha motor system. Arcln ,Ywrol. 6: 1044 I .3. Strahlendorf. J. C.. Strahlendorf. H. K., Kingsley, R. E.. Gmtautas, J. and Barnes, C. D. (19X0). Facilitation of the lumbar monosynapfic reflexes by locus coeruleus stimulation. h’r’urophu,rnuc~olog~ 19: 225- 230. Svensson, T. H. and Usdin. T. (1978). Feedback inhibltion of brain noradrenaline neurons by tricyclic antidepressants: m-receptor mediation. Science 202: 1089-1091. York, D. H. (1972). Potentiation of lumbo-sacral monosynaptic reflexes by the substantia nigra. Expl Neural. 36: 437-448. York, D. H. (1973). Antagonism of descending effects of the substantia nigra on lumbo-sacral monosynaptic reflexes. Nrurophnrmu~olo~l~ 12: 629-636.